The Leishmania spp. protozoa have a profound effect on the host cell that they invade. These parasites reside intracellularly usually in macrophages, a cell type with the capacity to kill intracellular microbes. Rather than succumb, Leishmania paralyze the microbicidal pathways of the host cell, changing the macrophage from a lethal cell to an intracellular safe haven which allows the parasite to survive, replicate and ultimately spread to neighboring cells. The mechanism(s) through which the parasite "paralyzes" the host microbicidal function is incompletely understood. It has recently come to light that most eukaryotic organisms utilize short noncoding RNA sequences to globally regulate expression of a wide variety of genes. Indeed short RNA sequences of 18-30 bp in length, called microRNAs, are critical for regulating expression of an estimated 30% of the human genome. The hypothesis underlying this proposal is that microRNAs are the upstream trigger(s) determining which pattern of macrophage activation will occur after Leishmania infection. We will address the following questions. (1) What microRNAs does the parasite usually induce or suppress during infection of macrophages? (2) Which microRNAs are induced in response to stimuli that activate macrophage toward different polar phenotypes? (3) Is there overlap between the microRNAs discovered in Aims 1 and 2, and what is the effect of experimental manipulation of these microRNAs on Leishmania-infected macrophages? Specific aims of the proposal are: 1. To perform a global profiling of changes in microRNA expression induced in response to phagocytosis of L. chagasi by human macrophages. 2. To use a similar profiling approach to determine which microRNAs are induced in response to macrophage activation toward different polarized phenotypes. Of primary interest will be the microRNA changes that occur during M1 (classical) activation with reciprocal changes in other forms of macrophage activation, since classically activated macrophages can kill intracellular Leishmania. 3. To correlate the results of Aims 1 and 2, and to selectively either overexpress or suppress macrophage expression of selected microRNAs that may change in the pattern of macrophage activation or intracellular parasite growth. PUBLIC HEALTH RELEVANCE: Leishmaniasis reached epidemic proportions during Operation Enduring Freedom (OEF) or Operation Iraqi Freedom (OIF), to a level not experienced by the US military since World War II. During 2002-03 there were more than 600 cases of cutaneous leishmaniasis and 4 cases of visceral leishmaniasis diagnosed in American soldiers deployed in Iraq, Kuwait and Afghanistan. Despite attention from international agencies, leishmaniasis remains a difficult disease to prevent and treat. The problem of leishmaniasis is becoming more prevalent in the VA system, as more troops return from stations in the Middle East. The purpose of this proposal is to explore the role of microRNAs in the pathogenesis of leishmaniasis, with the view that manipulation of small regulatory RNAs represents an innovative future avenue for therapy of this and other intracellular infectious diseases.